This is a new application for an R01 grant to establish the intellectual and technical framework to permit the study of viral infection on the single-cell level to be as tractable as the study of viral infection on the population level using the plaque assay. The world is ill equipped to deal with the (re)emergence of diseases caused by RNA viruses. The viral RNA genome is replicated by the virus-encoded RNA-dependent RNA polymerase (RdRp), an enzyme that, in most cases, lacks proofreading activity and a cellular repair mechanism to usurp. As a result, each progeny genome differs from another in the population by one or more nucleotide changes. The Cameron laboratory and colleagues have discovered that the genetic diversity created by replication errors made by the RdRp permits the virus to clear bottlenecks that would otherwise lead to viral extinction. Therefore, it is becoming increasingly clear that attenuated viruses for use as vaccine strains can be created by altering the nucleotide incorporation fidelity of the RdRp. Unexpectedly, the Cameron laboratory has observed that using conventional approaches to study this class of vaccine candidate in cell culture masks the attenuated phenotype. The attenuated phenotype is only observed by evaluating the infection at the level of the single cell instead of the population. This observatio motivated the development of techniques to study viral infection on the single-cell level. Our foray into this area was funded by the PSU Huck Institutes of the Life Sciences. In this proposal, we present a set of experimental objectives that will move single-cell virology from a descriptive art to a quantitative science that can be implemented broadly by the virology community not only to understand viral population dynamics but to reveal between-individual differences that may underlie susceptibility to viral infection. Importantly, this technology is essential to advancing RdRp fidelity as a target and mechanism for viral attenuation and vaccine development, thereby addressing an urgent public-health need. We will, therefore, pursue the following specific aims: (1) Elucidate parameters governing diverse kinetics of viral genome replication at the single-cell level; (2) Establish a data and statistical analysis pipeline for th single-cell virology experiment and develop mechanistic models of infection; and (3) Enhance capabilities of the microfluidic platform for characterization of viral infections at the single-cel level.